Physicochemical parameters, sensory profile and concentration of volatile compounds and anthocyanins in beers brewed using potato variety with purple flesh

In the recent years, beer brewers are experimenting with using various substrates, other than traditional barley malt, water, hops, and yeast for beer production, because new adjuncts to the beer brewing can add new sensory and functional properties to this beverage. Novel potatoes with purple or red-colour flesh are a good and cheap starch source and are rich in bioactive components, which could increase the nutritive value of the produced beer. The aim of the study was to determine whether some part of barley malt can be replaced by the potatoes of purple-colour flesh and assessment of properties of such beer. Beer samples showed increased antioxidant activity, higher concentration of anthocyanins and polyphenol compounds, as well as modified composition of volatiles and lower ethanol content. Beer produced with the addition of 30% of purple potatoes showed acceptable organoleptic qualities in the sensory analysis.

Biological material. Biological material used in this study was dried Saccharomyces cerevisiae yeast Fermentis US-05 from LeSaffre company (Marcq-en-Baroeul, France). Yeast was added to the cooled wort (20 °C) in the dose recommended by the producer (0.5 g d.m. per dm 3 of the wort). Twenty min prior to inoculation, yeast were rehydrated in sterile, distilled water at a temperature of 20 °C, in a ratio of 1 g d.m. of yeast per 20 cm 3 of water.
Brewing process. Beer brewing process was simultaneously performed three times, to prepare three different beers with various shares of the purple potatoes in the 'grain bill' of the brewed beer. Dry matter content of the potatoes compared to the extractivity of the malt was used to determine, how much of the potatoes must be used instead of the malt. The potatoes substituted malt in the ratio of 21.34:81. 21. In the first beer, the control sample (C), 100% of the extract in the 'grain bill' came from barley malt. In the second beer (P30), 30% of the assumed extract in the grain bill came from purple potatoes and 70% from the barley malt. In the third beer (P50), 50% of the assumed extract in the 'grain bill' came from the purple potatoes and 50% from the barley malt. 3 kg of malt were used to produce beer C; 2.1 kg of malt and 3.4 kg of peeled purple potatoes was used to brew beer P30; 1.5 kg of malt and 5.7 kg of peeled purple potatoes was used to brew beer P50. The purple potatoes, just prior the beer brewing process were peeled and cut into 1 cm thick slices. The slices were boiled in the 90 °C water for 20 min in the separate vessels for beer P30 and beer P50. After the boiling process, water was drained away from the boiled potatoes and potatoes were cooled to 20 °C and then mixed with barley malt (2.1 kg for P30, 1.5 kg for P50). From this point, the brewing was conducted with the same conditions for all the beers, in three different vessels. Barley malt or potatoes/malt mixtures were mixed with 15 kg of water with the temperature of 52 °C. The temperature of the mashes was adjusted to 52 °C and the mashing was conducted with the following time and temperature regime: • 10 min at 52 °C • 40 min at 62 °C • 20 min at 72 °C • 1 min at 78 °C Temperature increase between each of the steps was performed at a ratio of 1 °C per 1 min. After the mashing process, mashes were transferred to the filtration vessels and mashes were sparged with additional water with the temperature of 78 °C. The sparging and filtration process was conducted until the extract of wort leaving the filtration vessel was over 2% w/w, measured with handheld refractometer PAL-1 (Atago, Tokyo, Japan). The filtered worts were brought to the boiling point and hop pellets (3 g per dm 3 of the wort) were added to the worts, which were then boiled for 60 min. After the boiling process, worts were cooled to the temperature of 19 °C with the use of immersion chillers. The chilled worts were filtered and transferred to the disinfected 25 dm 3 fermenters. Rehydrated yeast was added to the chilled worts to start the process of fermentation and produce beer. Fermenters were kept for 14 days in the refrigerated cabinets with temperature set at 19 °C. After the fermentation process, beer were transferred from the yeast beds on the bottom of the fermenters and bottled with the addition of 6 g of glucose per dm 3  www.nature.com/scientificreports/ energy content were measured using Anton Paar DMA 4500 densimeter and alcolyzer (Graz, Austria). Beer pH was analysed with the use Mettler Toledo MP 240 pH-meter (Columbus, DC, USA). Beer samples, prior to the analyses were degassed, mixed with diatomaceous earth (1 g per 100 cm 3 of beer) and filtered through the laboratory filter paper. Six analyses were performed for each of the beers (two repetitions from three different bottles of the same beer).
Volatile compounds. Volatile compounds of the tested beers were analysed by the gas chromatography technique coupled with flame ionizing detection (GC-FID), using a GC2010 Plus apparatus with a FID-2010 and a headspace autosampler (HS-20) (Shimadzu Corporation, Kyoto, Japan), equipped with a CP-WAX 57 CB column (50 m × 0.32 mm ID × 0.2 µm) (Agilent Technologies, Santa Clara, CA, USA). Beer samples were degassed, mixed with diatomaceous earth (1 g per 100 cm 3 of beer), and filtered through a paper filter. After filtration, 10 cm 3 of beer was transferred to a 20 cm 3 headspace vial. Each vial was conditioned in a headspace autosampler oven set at 40 °C and equilibrated for 20 min at shaking level 2 prior to the injection of the sample into the column. The operating conditions of the chromatographic analysis are described in detail in the work published by Kawa-Rygielska et al. 8 Six measurements were performed for each of the beers (two repetitions from three different bottles of the same beer).
Carbohydrate profile and concentration of glycerol. The concentration of carbohydrates and glycerol were examined by the means of high-performance liquid chromatography (HPLC). Beer samples were degassed, centrifuged (6000 rpm, 10 min), diluted (1:1) with ultrapure water and filtered through syringe nylon filters (0.45 µm pore size) to chromatographic vials. The samples were then analyzed using a Prominence liquid chromatography system (Shimadzu Corp., Kyoto, Japan) equipped with a Rezex ROA-Organic Acid H + column (300 × 4.6 mm) from Phenomex (Torrance, CA, USA). The parameters of the measurements were previously described in the work of Pietrzak et al. 10 Six analyses were performed for each of the beers (two repetitions from three different bottles of the same beer).
Concentration of phenolic compounds. Concentration of phenolic compounds of the beers was assessed using the Folin-Ciocalteu spectrophotometric method customized for beer, previously described by Kawa-Rygielska et al. 11 Data were expressed as mg of gallic acid equivalent per dm 3 of beer (GAE/dm 3 ). Calibration curve (R value = 0.998) in the range of 0.30-9.00 mg GAE/cm 3 was used to read the results. Nine analyses were performed for each of the beers (three repetitions from three different bottles of the same beer).
Free radical scavenging ability (ABTS ·+ assay). The antiradical ability of the beers was determined using ABTS •+ assay customized for beer, described previously in the work by Kawa-Rygielska et al. 11 The data were expressed as Trolox equivalent (TE) of antioxidative capacity per dm 3 of the beer (mmol TE/dm 3 ). Calibration curves, in the range 1.7-21.7 µmol TE/cm 3 , showing good linearity (r 2 ≥ 0.999) were prepared using Trolox solution (0.005 mmol/dm 3 ). Nine analyses were performed for each of the beers (three repetitions from three different bottles of the same beer).
Free-radical scavenging ability (DPPH· assay). The antiradical ability of the beers was determined using DPPH• assay customized for beer, described previously in the work by Kawa-Rygielska et al. 11 The data were expressed as Trolox equivalent (TE) of antioxidative capacity per dm 3 of the beer (mmol TE/dm 3 ). Calibration curves, in the range 2-10 µmol TE/cm 3 , showing good linearity (r 2 ≥ 0.998) were prepared using Trolox solution (0.005 mmol/ dm 3 ). Nine analyses were performed for each of the beers (three repetitions from three different bottles of the same beer).
Ferric reducing (antioxidant) power (FRAP assay). The antioxidant power of the beers was determined using FRAP assay customized for beer, described previously in the work by Kawa-Rygielska et al. 11 The data were expressed as Trolox equivalent (TE) of antioxidant power per dm 3  Statistics. Data were processed using Statistica 13.5 software (Statsoft, Tulsa, OK, USA) using one way ANOVA (α = 0.05). Duncan test was used to analyse differences between means (p < 0.05) and to establish homogenous groups.

Results and discussion
Beer physicochemical parameters. Beers brewed with the use of purple potatoes were characterised with slightly different basic physicochemical parameters, shown in the Table 1.
Extract of the wort was lower for beers P30 and P50, which would be in accordance with the, for example lower glycerol content as well as lower amylase activity mentioned already in the Section "Concentration of anthocyanins and phenolic acids". Attenuation of the beer, beer alcohol content as well as real extract of the beer further confirm the fact, that during the mashing more of the unfermentable sugars were produced in the beers P30 and P50 2 . Lower energy content is a consequence of the lower alcohol content of the beers brewed with the use of purple potato, as the ethanol is the main source of energy in beer and most of the alcoholic beverages. 12,13 Lower pH of the beer C can also be easily explained by the lower alcohol content of the finished beer, as the beer pH decreases during the fermentation 2 . Typically beers with higher wort extract content ought to be darker (and acquire higher EBC parameter) due to the extraction of Maillard reaction type compounds from the malt 2 , but samples P30 and P50 were characterised with higher EBC parameter than the C. The concentration of anthocyanins is probably the main factor of the EBC parameter increase, as the sample C, characterised with the lowest EBC value possessed none of the anthocyanins and the EBC is the highest for P50, in which content of anthocyanins is the highest. The study published by Moirangathen et al. 14 about the beer brewed with the use of black rice, in which they assessed the content of anthocyanins, acquired the same results: beers with highest anthocyanin content had highest EBC value.
Volatile compounds. GC-FID analysis allowed for identification and quantification of 21 volatile components (8 alcohols, 4 aldehydes, 5 esters and 4 pyrazines) out of 30 analysed, which are shown in Table 2.
Compounds such as izobutyraldehyde, butanal, 3-methylbutanal, methanol, 2,3-butanodione, ethyl heptanoate and 1-hexanol were not detected in the analysed beers. Beers brewed with the addition of potato were characterised with far lower total concentration of volatiles (182.39 mg/dm 3 in P50 and 222.59 mg/dm 3 in P30) than C (320.74 mg/dm 3 ), albeit higher concentration of volatile components does not have to be an advantage, as many volatiles in the alcoholic beverages possess undesirable aroma, when they are present in the beer in great quantity 15 . Many of the so-called 'higher alcohols' or 'fusel alcohols' , such as 1-propanol, 2-methylbutanol, 3-methylbutanol, 1-pentanol, phenylethyl alcohol and isobutanol belong to this category 16 . In the analysed beers an interesting influence of substituting barley malt with potatoes on the concentration of fusel alcohols can be seen. Higher percentage of potatoes in the 'grain bill' of the brewed beer resulted in lower concentration of 2-methylbutanol, 3-methylbutanol and phenylethyl alcohol, but concentration of 1-propanol and 1-butanol increased. Decrease in the concentration of 2-methylbutanol and 3-methylbutanol, as well as phenylethyl alcohol can be a result of far lower concentration of amino acids in the wort because Saccharomyces cerevisiae yeast produces these alcohols by the Ehrlich pathway, from the leucine, isoleucine and phenylalanine 16 . As the potatoes (typical potatoes and potatoes with coloured-flesh alike) possess lower concentration of these amino acids than barley, this explanation seems most plausible 17,18 . Different result can be seen in the concentration of 1-propanol. Samples P30 and P50 are characterised with higher concentration of this alcohol than the sample C. This alcohol is produced by the Saccharomyces cerevisiae yeast from amino acid threonine 19 . As the potatoes are not far richer in threonine than barley, then it can be assumed that during conditions of mashing, during which not all the proteins present in the malt are hydrolysed 2 , more threonine from the potatoes was released to the wort than from the barley malt. It is also interesting to note, that beers brewed with the addition of potato www.nature.com/scientificreports/ tubers were not characterised with methanol content. Typically, methanol is present in small amounts in various alcoholic beverages produced from the fruit, as the fruits can be characterised with the presence of small quantity of this alcohol and, more importantly, are source of pectins 20 . Pectins can be dagreded by pectinases produced by the Saccharomyces cerevisiae yeast, resulting in formation of minuscule amount of ethanol 20 . This is why, typically, beer posseses virtually none of methanol, in comparison to the beverages created from fruit musts, such as wines or wine distillates, as the barley malt is not a good source of pectins. On the other hand, potato tubers, in contrast to barley, are a rich source of various pectins, which should suggest, that beers brewed with the addition of potatoes should be characterised with methanol content 21 . However, beer-making process uses prolonged time of heat treatment during the mashing and wort boiling procedures and pectins are degraded by various thermal treatments, which means that in the resulting wort the concentration of pectins was too low to produce significant amounts of methanol 2, 22 . The influence of addition of purple potato on two more compounds, very important in the beer brewing industry, should be closely analysed. Acetaldehyde is an intermediate in the production of many volatile compounds during the process of yeast fermentation, such as acetic acid (during the generation of acetyl-CoA and acetate esters) as well as ethanol, which is why the presence of this compound is rather unavoidable during the beer production. In low concentrations, this compounds is related to the pleasant green apple aroma, but higher content significantly worsens beverage flavour 23 . All the produced beers were characterised with the concentration of acetaldehyde in which it is not unpleasant and irritating 24 . However, it is worth to mention that the influence of potato addition to the produced beer resulted in far lower concentration of acetaldehyde than in control sample. P30 contained 45.72% of the acetaldehyde detected in C, while P50 contained just 25.67% of this volatile. This result should be seen as the advantage of using purple potatoes in the beer production technology, as acetaldehyde is far more toxic than the ethanol present in alcoholic beverages such as beer and wine 25,26 . Lower concentration of ethyl acetate, compound produced by yeast from glucose, might be the result of lower concentration of fermentable sugars in the worts from which the beers P30 and P50 were produced. High pressure liquid chromatography of beers analysed in this study confirmed, that beers P30 and P50 possess higher concentration of dextrins (which are unfermentable by Saccharomyces cerevisiae) and lower concentration of maltose and glucose 2 .

Concentration of anthocyanins and phenolic acids. Beers produced with the use of purple potatoes
were characterised with concentration of various anthocyanins and phenolic acids (shown in Table 3), which were absent in the control sample, which shows that only potatoes were source of these compounds. Four anthocyanins and three phenolic acids were identified and quantified in the samples P30 and P50. Total concentration of anthocyanins in P30 was equal to 12.383 mg per dm 3 and in P50 equal to 36.507 mg per dm 3 of beer. The most abundant anthocyanin in both beer samples was petunidin 3-(p-coumaroyl)-rutinoside-5-glucoside and its concentration exceeded 50% of total anthocyanin content. Concentration of these substances in potatoes of purple-coloured flesh is usually far higher, ranging for substances such as petunidin  Three phenolic acids, such as chlorogenic acid, cryptochlorogenic acid and neochlorogenic acid were identified in P30 and P50. Concentration of these compounds was far greater than concentration of anthocyanins and was equal to 360.507 mg for P30 and 721.555 mg for P50. Such great difference between the concentration of phenolic acids and anthocyanins in the beer ought not to be surprising, as the coloured potatoes possess significantly higher concentration of chlorogenic, neochlorogenic and cryptochlorogenic acid than anthocyanins 33,34 . Additionally, these three acids are characterised with high stability during the heat treatment and in the low pH environment which easily explains higher concentrations of these particular chemicals in the prepared beverages 35,36 . Table 4 shows concentration of carbohydrates (glucose, maltose, maltotriose and dextrinsand glycerol in beers brewed with the use of potatoes with purple-coloured flesh. Beers P30 and P50 were characterised with higher concentration of unfermentable dextrins, with P50 possessing more dextrins than P30, which suggests, that the starch present in potatoes hydrolyses into less fermentable sugars than barley starch, albeit it might be also a result of lack of proper concentration of beta-amylase enzyme, which cleaves maltose from the starch. With lower amount of barley malt in the 'grain bill' of the produced beer, concentration of amylases in the mash decreased, which might result in inadequate activity of amylases during the process of beer production. Therefore, in the beers P30 and P50 more time would be needed during mashing at the temperature of 62 °C (which is optimal for beta-amylase activity) to produce as much fermentable sugars as in the sample C 2 . Additionally, lower concentration of fermentable sugars in the worts used to produce beers P30 and P50 would also explain lower concentration of glycerol in the analysed samples, because glycerol is produced by the Saccharomyces cerevisiae yeast as the by-product of ethanol fermentation 37,38 . Table 3. Concentration of anthocyanins and phenolic acids in beers produced with the use of violet potatoes. 1 Values are expressed as a mean (n = 6) ± standard deviation. Mean values with different letters (a, b) within the same row are statistically different (α = 0.05) according to Duncan test. n.d.-compound not detected. www.nature.com/scientificreports/ Concentration of phenolic compounds and antioxidant activity of the beers. The total phenolic content of the beer as well as beer antioxidant activity is shown in the Table 5.

Concentration of carbohydrates and glycerol.
The use of potatoes with purple-coloured flesh increased concentration of phenolic compounds in the beers, as well as the antioxidant activity of the beverages. As the main phenolic compounds in the beers originate from barley, which is not especially rich in phenolic compounds, changing part of the 'grain bill' of the beer for potatoes with purple-coloured flesh, which have been proven to possess high concentration of phenolic compounds, as well as high antioxidant activity (Rytel et 34 ) ought to increase phenolic compound content, as well as increase beer antioxidant activity, which is confirmed by the results acquired in this study 2,31,39-41 . The increased concentration of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid, as well as anthocyanins is probably the main reason of the increased activity of the P30 and P50 beers, because these compounds have been proven in the past to be strong antioxidants 42,43 . Sensory analysis. The results of the sensory analysis ( Table 6) have shown that P50 is inferior to the P30 and C beer in most of the assessed parameters.
Nevertheless, P50 still acquired high score (6.20) for the colour parameters. P30 and C were similarly rated in all of the tested parameters, with P30 acquiring slightly lower score in the taste parameter. Panellists described beer P50 as one, which had 'foreign' taste and smell and 'unpleasant' aftertaste. These results show that 30% of extract in the brewed beer is the appropriate dosage of potatoes which can be easily used without need of any further changes in the beer recipe to acquire palatable beverage. Still, it is important to mention, that addition of unusual substrates into the beer production often results in changing sensory attributes of the beer, however, by applying appropriate changes into the beer production regime, most of the disadvantages can be mitigated 2 . For example, studies of Vrzal et al. (2021) have shown that changes in the amino acids content in the finished beer correspond with changes in sensory properties of the beer in the aspects of beer 'fullness' , 'astringency' , 'sourness' or 'sweetness' , therefore by applying changes to the protein rest during the mashing procedure, beer brewers could influence aroma of the beer brewed with addition of potatoes 44 . Similarly, various malt additives, such as dark malts or caramel malts could be used during the beer brewing to change taste and aroma of the produced beverage 45 . Additional method, which is gaining popularity nowadays, is using unconventional yeast or other microorganisms (such as lactic acid bacteria) during the process of beer fermentation to modify taste, aroma, attenuation degree or even colour of the produced beer 46 . Beer taste, presence and aroma can be also modified in great extent by addition of various hops and fruits 46,47 .

Conclusions
Results acquired in this study show, that there is a viable possibility of using potatoes with purple-coloured flesh in the beer brewing technology. Produced beers acquired rather satisfactory physicochemical properties important in the beer production. Additionally, brewing beer with the addition of potatoes with purple-coloured flesh resulted in beers with higher concentration of polyphenolic compounds, higher antioxidant activity and lower calorie value and alcohol content. Analysis of volatile components in the beers brewed with the use of potatoes with purple-coloured flesh showed that these beers possess the whole range of compounds characteristic for pilsner-type beers, but usually in lower quantities than in control beer. Further studies on the beers brewed with Table 5. Total phenolic content (TPC) and antioxidant activity (ABTS, DPPH, FRAP) of the beers produced with the use of purple potatoes. 1 Values are expressed as a mean (n = 9) ± standard deviation. Mean values with different letters (a, b) within the same row are statistically different (α = 0.05) according to Duncan test. 2 GAE-gallic acid equivalent. 3  www.nature.com/scientificreports/ addition of potatoes could be concentrated on the adjustment of the beer brewing conditions to combine novel pro-health benefits of compounds with acceptable organoleptic quality of finished product.

Data availability
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.